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R Reeves

Publications and source records attributed to R Reeves.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of the proto-oncogene pim-1: kinase activity and substrate recognition sequence.

The human pim-1 proto-oncogene was expressed in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein and the enzymatic properties of its kinase activity were characterized. Likewise, a Pim-1 mutant lacking intrinsic kinase activity was constructed by site-directed mutagenesis (Lys67 to Met) and expressed in E. coli. In vitro assays with the mutant Pim-1 kinase showed no contaminating kinase activity. The wild-type Pim-1 kinase-GST fusion protein showed a pH optimum of 7 to 7.5 and optimal activity was observed at either 10 mM MgCl2 or 5 mM MnCl2. Higher cation concentrations were inhibitory, as was the addition of NaCl to the assays. Previous work by this laboratory assaying several proteins and peptides showed histone H1 and the peptide Kemptide to be efficiently phosphorylated by recombinant Pim-1 kinase. Here we examine the substrate sequence specificity of Pim-1 kinase in detail. Comparison of different synthetic peptide substrates showed Pim-1 to have a strong substrate preference for the peptide Lys-Arg-Arg-Ala-Ser*-Gly-Pro with an almost sixfold higher specificity constant kcat/Km over that of the substrate Kemptide (Leu-Arg-Arg-Ala-Ser*-Leu-Gly). The presence of basic amino acid residues on the amino terminal side of the target Ser/Thr was shown to be essential for peptide substrate recognition. Furthermore, phosphopeptide analysis of calf thymus histone H1 phosphorylated in vitro by Pim-1 kinase resulted in fragments containing sequences similar to that of the preferred synthetic substrate peptide shown above. Therefore, under optimized in vitro conditions, the substrate recognition sequence for Pim-1 kinase is (Arg/Lys)3-X-Ser/Thr*-X', where X' is likely neither a basic nor a large hydrophobic residue.

Amino Acid Sequence↗
PubMed1992

Constitutive IL-2 mRNA expression in lymphocytes, infected with the intracellular parasite Theileria parva.

Theileria parva-infected lymphoblastoid cell lines of T or B cell origin were examined for IL-2 mRNA expression. T. parva-infected T cell lines could be of the CD4-CD8-, CD4+CD8-, CD4-CD8+, or CD4+CD8+ phenotype and express alpha beta or gamma delta TCR. By Northern blot analysis and amplification by the polymerase chain reaction, IL-2 mRNA could be detected in all T. parva-infected cell lines tested. IL-2 mRNA expression was also shown to be dependent on the continuous presence of the parasite in the host cell cytoplasm, because elimination of the parasite by treatment of T. parva-infected cell cultures with the theilericidal drug BW720c resulted in the disappearance of detectable IL-2 mRNA. The effect of anti-IL-2 antibodies on the proliferation of T. parva-infected cells was also tested. Inhibition experiments suggest that although IL-2 mRNA can be detected in all cell lines tested, not all T. parva-infected cell lines are dependent on IL-2 for their proliferation. Our data provide the first example for the constitutive expression of IL-2 mRNA in T and B cells caused by infection with an intracellular parasite.

Animals↗
PubMed1992

Characterization of the testes-specific pim-1 transcript in rat.

The pim-1 proto-oncogene encodes a serine/threonine protein kinase and is expressed in cells of hematolymphoid origin and in the germ cell lineages. In somatic cells, the pim-1 gene is expressed as a 2.8 kb transcript while a shorter sized transcript (2.3 kb) is expressed in rat testes. We have determined that the shorter testes-specific pim-1 transcript arises through the use of an alternate polyadenylation signal present in the 3' untranslated region of the gene. This alternate polyadenylation event results in the removal of an A/U-rich regulatory element located in the 3' untranslated region of the pim-1 gene. This A/U-rich motif has been shown by a number of laboratories to destabilize the transcripts of genes that contain this sequence. Consistent with these findings, we have demonstrated that the shortened testes-specific pim-1 transcript is more stable than the longer A/U-rich containing somatic transcript. We suggest that the functional significance of different sized pim-1 transcripts may be directly related to their different stabilities and that the greater stability of the testes-specific transcript may be essential for the translational delay observed in post-meiotic male germ cells.

Amino Acid Sequence↗
PubMed1992

Hoechst 33258, distamycin A, and high mobility group protein I (HMG-I) compete for binding to mouse satellite DNA.

The experiments described were designed to test the hypothesis that the (A+T)-specific DNA binding ligands Hoechst 33258 and distamycin A affect the condensation of mouse centromeric heterochromatin by competing for binding to satellite DNA with one or more chromosomal proteins. The studies focused on the nonhistone chromosomal protein HMG-I since its binding properties predict it would be a target for competition. Gel mobility shift assays show that HMG-I forms specific complexes with satellite DNA and that the formation of these complexes is competed for by both Hoechst and distamycin. In addition, methidium propyl EDTA Fe(II) [MPE Fe(II)] footprints of ligand-satellite DNA complexes showed essentially the same protection pattern for both drugs and a similar, but not identical, HMG-I footprint. If these in vitro results reflect the in vivo situation then the incomplete condensation of centromeric heterochromatin observed when mouse cells are grown in the presence of either chemical ligand could be a consequence of competition for binding of HMG-I (and possibly other proteins) to satellite DNA.

Animals↗
PubMed1992

Chromatin changes during the cell cycle.

Considerable progress has recently been made in elucidating the biochemical mechanisms regulating changes in chromatin structure during all stages of the cell cycle. Although anticipated, the apparently ubiquitous role played by phosphorylation/dephosphorylation reactions in modulating these changes is, nonetheless, remarkable.

Animals↗
PubMed1992

A poly(dA-dT) upstream activating sequence binds high-mobility group I protein and contributes to lymphotoxin (tumor necrosis factor-beta) gene regulation.

Lymphotoxin (LT; also known as tumor necrosis factor-beta) is a pleiotropic cytokine whose expression is tightly regulated in most cells and is repressed prior to activation signals. In some early B cells and Abelson murine leukemia virus-transformed pre-B-cell lines, LT mRNA is constitutively expressed. To examine the molecular regulation of the LT gene in a constitutively expressing cell line, we studied the Abelson murine leukemia virus-transformed lines PD and PD31. As demonstrated by primer extension analysis, constitutively expressed pre-B-cell-derived and inducibly expressed T-cell-derived LT mRNA were initiated at the same cap sites and predominant cap site utilization was conserved. Furthermore, we delineated an upstream activating sequence that was an important functional component of lymphotoxin transcriptional activation in PD and PD31 cells. The upstream activating sequence was localized to an essentially homopolymeric A + T-rich region (LT-612/-580), which was bound specifically by recombinant human high-mobility group I protein (HMG-I) and a PD/PD31 nuclear extract HMG-I (HMG-I-like) protein. The nuclear extract-derived HMG-I-like protein was recognized by anti-HMG-I antibody and bound to LT DNA to effect an electrophoretic mobility shift identical to that of bound recombinant human HMG-I. These findings implicate HMG-I in the regulation of constitutive lymphotoxin gene expression in PD and PD31 cells. HMG-I and HMG-I-like proteins could facilitate the formation of active initiation complexes by altering chromatin structure and/or by creating recognition sites for other activator DNA-binding proteins, some of which may be unique to or uniquely modified in these constitutive LT mRNA producers.

Base Sequence↗
PubMed1991

Stability changes in pim-1 proto-oncogene mRNA after mitogen stimulation of normal lymphocytes.

mRNA expression of the serine/threonine kinase protooncogene Pim-1 was investigated in mitogen-treated normal bovine lymphocytes. After stimulation with Con A and phorbol ester (PMA), normal bovine PBMC exhibited a 3.5-fold induction of pim-1 mRNA within 4 h of stimulation. By 17 h poststimulation, however, the level of pim-1 mRNA had fallen to approximately 50%. Similar transient kinetics of pim-1 expression were also observed in mitogen-stimulated bovine lymph node lymphocytes and the amount of pim-1 mRNA induced was dependent on the type of mitogen stimulus. Typically, stimulation with Con A and PMA together acted synergistically resulting in a greatly increased amount of pim-1 mRNA induction compared to stimulation with either Con A or PMA alone. To determine if an increase in pim-1 mRNA stability contributed to the overall increase in pim-1 mRNA levels observed after mitogen stimulation, RNA half-life studies were performed. At 4 h poststimulation, pim-1 mRNA in bovine lymph node lymphocytes was relatively stable with a t1/2 of approximately 80 min; at 17 h poststimulation, however, the t1/2 (approximately 35 min) was markedly decreased. In addition, cyclohexamide treatment was found to markedly increase the stability of pim-1 transcripts in bovine PBMC suggesting that a protein synthesis-dependent posttranscriptional pim-1 mRNA degradation pathway may be involved in the regulation of pim-1 mRNA levels in lymphoid cells. To investigate the possible contribution of the destabilizing (UAUU)n motif to pim-1 mRNA stability, the t1/2 of the short 2.4-kb germ cell-specific pim-1 transcript found in rat testes was compared to that of the longer 2.8-kb somatic transcript expressed in stimulated rat lymphocytes. t1/2 determination experiments showed that the 2.4-kb testes-specific transcript, which is missing the destabilizing (UAUU)n sequence, was quite stable (t1/2 much greater than 6 h). In contrast, the t1/2 of the longer 2.8 kb somatic cell pim-1 transcript that contains this A/U-rich sequence motif was found to be much shorter (t1/2 approximately 130 min) in mitogen-activated rat lymphocytes. Together these findings indicate that the transient induction of pim-1 gene expression is associated with normal lymphocyte activation and that the stability of pim-1 transcripts is regulated in lymphocytes during the course of activation. In addition, the differences in pim-1 mRNA stability observed in germ cells and lymphocytes of the same animal are likely the result of molecular mechanisms involving differential regulation by the message destabilizing (UAUU)n motif.

Animals↗
PubMed1991

Phosphorylation by cdc2 kinase modulates DNA binding activity of high mobility group I nonhistone chromatin protein.

Chromatin high mobility group protein I (HMG-I) is a mammalian nonhistone protein that has been demonstrated both in vitro and in vivo to preferentially bind to A.T-rich sequences of DNA. Recently the DNA-binding domain peptide that specifically mediates the in vitro interaction of high mobility group protein (HMG)-I with the narrow minor groove of A.T-DNA has been experimentally determined. Because of its predicted secondary structure, the binding domain peptide has been called "the A.T hook" motif. Previously we demonstrated that the A.T hook of murine HMG-I protein is specifically phosphorylated by purified mammalian cdc2 kinase in vitro and that the same site(s) are also phosphorylated in vivo in metaphase-arrested cells. We also found that the DNA binding affinity of short synthetic binding domain peptides phosphorylated in vitro by cdc2 kinase was significantly reduced compared with unphosphorylated peptides. Here we extend these findings to intact natural and recombinant HMG-I proteins. We report that the affinity of binding of full-length HMG-I proteins to A.T-rich sequences is highly dependent on ionic conditions and that phosphorylation of intact proteins by cdc2 kinase reduces their affinity of in vitro binding to A.T-DNA by about 20-fold when assayed near normal mammalian physiological salt concentrations. Furthermore, in cell synchronization studies, we demonstrated that murine HMG-I proteins are phosphorylated in vivo in a cell cycle-dependent manner on the same amino acid residues modified by purified cdc2 kinase in vitro. Together these results strongly support the assertion that HMG-I proteins are natural substrates for mammalian cdc2 kinase in vivo and that their cell cycle-dependent phosphorylation by this enzyme(s) significantly modulates their DNA binding affinity, thereby possibly altering their biological function(s).

Amino Acid Sequence↗
PubMed1991

Recombination of exogenous interleukin 2 receptor gene flanked by immunoglobulin recombination signal sequences in a pre-B cell line and transgenic mice.

We have constructed a plasmid, pLTR100, which contains human interleukin 2 receptor light (IL-2R L) chain cDNA in the inverted orientation relative to the upstream SV40 promoter. The cDNA segment is flanked by the immunoglobulin gene recombination signal sequences so that the cDNA segment can invert and the human IL-2R L chain is subsequently expressed under the control of the SV40 promoter. A murine pre-B cell line, 38B9, transfected with pLTR100 began to express the human IL-2R L chain on the cell surface. The frequency of human IL-2R L chain positive cells increased almost linearly up to 50% for 60 days of culture after transfection. Southern blot analysis and sequencing of the DNA fragments at the recombination junction confirmed that the cDNA segment was inverted in a signal sequence-dependent manner by the variable-diversity-joining recombination process. Transgenic mice bearing the recombination substrate DNA similar to pLTR100 expressed the human IL-2 L chain in the spleen, thymus, and bone marrow, but not in the other tissues examined at the detectable level. Both IgM- and CD3-positive cells expressed the human IL-2R L chain, indicating that this artificial DNA can serve as a substrate for recombination both in B- and T-cells and that another DNA segment may be necessary to confer the cell-type specificity on the substrate DNA.

Animals↗
PubMed1991

Recombinant human pim-1 protein exhibits serine/threonine kinase activity.

The protein predicted by the sequence of the human pim-1 proto-oncogene shares extensive homology with known serine/threonine protein kinases, and yet the human Pim-1 enzyme has previously been reported to exhibit protein tyrosine kinase activity both in vitro and in vivo. Recently a new class of protein kinases has been identified which exhibits both protein-serine/threonine and protein-tyrosine kinase activities. We therefore investigated the possibility that the human Pim-1 kinase likewise possesses such bifunctional enzymatic phosphorylating activities. A full-length human pim-1 cDNA was subcloned into the bacterial vector pGEX-2T and the Pim-1 protein expressed as a fusion product with bacterial glutathione S-transferase (GST). The hybrid GST-Pim-1 fusion protein was affinity purified on a glutathione-Sepharose column prior to treatment with thrombin for cleavage of the Pim-1 protein from the transferase. Pim-1 was purified and the identity of recombinant protein confirmed by amino-terminal sequence analysis. Pim-1 was tested for kinase activity with a variety of proteins and peptides known to be substrates for either mammalian protein-serine/threonine or protein-tyrosine kinases and was found to phosphorylate serine/threonine residues exclusively in vitro. Both the Pim-1-GST fusion protein and the isolated Pim-1 protein exhibited only serine/threonine phosphorylating activity under all in vitro conditions tested. Pim-1 phosphorylated purified mammalian histone H1 with a Km of approximately 51 microM. Additionally, Pim-1 exhibited low levels of serine/threonine autophosphorylating activity. These observations place the human Pim-1 in a small select group of cytoplasmic transforming oncogenic kinases, including the protein kinase C, the Raf/Mil, and the Mos subfamilies, exhibiting serine/threonine phosphorylating activity.

Cloning, Molecular↗
PubMed1991

Phosphorylation of the DNA-binding domain of nonhistone high-mobility group I protein by cdc2 kinase: reduction of binding affinity.

Mammalian high-mobility group I nonhistone protein (HMG-I) is a DNA-binding chromatin protein that has been demonstrated both in vitro and in vivo to be localized to the A + T-rich sequences of DNA. Recently an unusual binding domain peptide, "the A.T-hook" motif, that mediates specific interaction of HMG-I with the minor groove of DNA in vitro has been described. Inspection of the A.T-hook region of the binding domain showed that it matches the consensus sequence for phosphorylation by cdc2 kinase. Here we demonstrate that HMG-I is a substrate for phosphorylation by purified mammalian cdc2 kinase in vitro. The site of phosphorylation by this enzyme is a threonine residue at the amino-terminal end of the principal binding-domain region of the protein. Labeling of mitotically blocked mouse cells with [32P]phosphate demonstrates that this same threonine residue in HMG-I is also preferentially phosphorylated in vivo. Competition binding studies show that cdc2 phosphorylation of a synthetic binding-domain peptide significantly weakens its interaction with A + T-rich DNA in vitro, and a similar weakening of DNA binding has been observed for intact murine HMG-I protein phosphorylated by the kinase in vitro. These findings indicate that cdc2 phosphorylation may significantly alter the DNA-binding properties of the HMG-I proteins. Because many cdc2 substrates are DNA-binding proteins, these results further suggest that alteration of the DNA-binding affinity of a variety of proteins is an important general component of the mechanism by which cdc2 kinase regulates cell cycle progression.

Amino Acid Sequence↗
PubMed1991

Influence of short term dietary cholesterol and fat on human plasma Lp[a] and LDL levels.

The relationship between plasma levels of Lp[a] and LDL was examined using dietary regimens. In 81 normolipidemic male outpatients, dietary cholesterol was increased by consuming six eggs per day from a mean (SD) level of 311 (162) to 1430 (198) mg per day. Mean (SD) LDL-cholesterol levels increased from 102 (26) mg/dl to 120 (33) mg/dl (P less than 0.001), while mean (SD) Lp[a] levels were 5.5 (6.1) mg/dl on the basal diet and 5.6 (6.4) mg/dl on the cholesterol-rich diet. No significant correlation was observed between increases in either LDL-cholesterol or apolipoprotein B to Lp[a], nor was there any relationship between individual baseline levels of Lp[a] and dietary-induced changes of Lp[a]. Fourteen of the 81 participants were reexamined under strict nutritional control. Four diets with 40% of calories as fat, but differing in the type of fat and the amount of cholesterol, were administered sequentially to all subjects. As expected, mean (SD) LDL-cholesterol and apolipoprotein B levels were highest on the saturated fat, high cholesterol diet (112 (32) mg/dl and 79 (22) mg/dl) and lowest on the polyunsaturated fat, low cholesterol diet (77 (27) mg/dl and 53 (18) mg/dl). In contrast, mean Lp[a] levels did not significantly change among the four diets (range 4.2-4.9 mg/dl). No correlation of Lp[a] responses with changes in plasma lipids, apolipoproteins, or lipoproteins was observed on any diet. These data suggest that determinants of plasma Lp[a] levels are distinctly different from the determinants of plasma LDL levels in normolipidemic males.

Adult↗
PubMed1990

Primary structure of the putative human oncogene, pim-1.

A full-length (6.1-kb) human genomic pim-1 gene, together with its immediate 5'-upstream promoter sequence (Ppim) was isolated and sequenced. The human pim-1 gene shares an overall nucleotide (nt) sequence identity of 53% with the previously reported murine pim-1 gene. It consists of six exons and five introns and contains a protein-coding region that is identical in nt sequence to a full-length human pim-1 cDNA. The gene codes for a predicted Pim-1 protein of 313 amino acids (aa) with an Mr of 35,690 and a pI of 5.7. The deduced aa sequence of the human Pim-1 has 94% identity with the murine Pim-1 whereas the nt sequences of the two genes are 88% identical. All of the conserved aa residues of the mouse pim-1 gene, which are homologous to known protein kinases are conserved in the predicted human protein. The human Ppim region is very G + C-rich (69%) and shares greater than 80% identity with the murine Ppim. The Ppim has no TATA- or CAAT-box sequences but does contain a number of nt sequences similar to the putative binding sites of several presumptive transcription factors.

Amino Acid Sequence↗
PubMed1990

The A.T-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure.

We have determined the domains of the mammalian high mobility group (HMG)I chromosomal proteins necessary and sufficient for binding to the narrow minor groove of stretches of A.T-rich DNA. Three highly conserved regions within each of the known HMG-I proteins is closely related to the consensus sequence T-P-K-R-P-R-G-R-P-K-K. A synthetic oligopeptide corresponding to this consensus "binding domain" (BD) sequence specifically binds to substrate DNA in a manner similar to the intact HMG-I proteins. Molecular Corey-Pauling-Koltun model building and computer simulations employing energy minimization programs to predict structure suggest that the consensus BD peptide has a secondary structure similar to the antitumor and antiviral drugs netropsin and distamycin and to the dye Hoechst 33258. In vitro these ligands, which also preferentially bind to A.T-rich DNA, have been demonstrated to effectively compete with both the BD peptide and the HMG-I proteins for DNA binding. The BD peptide also contains novel structural features such as a predicted Asx bend or "hook" at its amino-terminal end and laterally projecting cationic Arg/Lys side chains or "bristles" which may contribute to the binding properties of the HMG-I proteins. The predicted BD peptide structure, which we refer to as the "A.T-hook," represents a previously undescribed DNA-binding motif capable of binding to the minor groove of stretches of A.T base pairs.

Adenine↗
PubMed1990

Developmental expression of somatostatin in mouse brain. II. In situ hybridization.

The distribution and the levels of expression of preprosomatostatin (PPSOM) mRNA were examined during pre- and postnatal development of the mouse brain using the in situ hybridization technique. The signal obtained by in situ hybridization of embryonic tissues at day 14 and day 17 of gestation was highest over the neurons of the pyriform cortex, amygdala, and entopeduncular nucleus. The signal was very low over cells of the neocortex and the developing hippocampal formation. The density of grains overlying the neurons of the amygdala and pyriform cortex continued to be high during early postnatal life, but decreased as the animals became adults. A progressive increase of PPSOM mRNA expression was observed in postnatal animals in the stratum oriens and dentate gyrus of the hippocampal formation. In the cerebral cortex and striatum, the number of these neurons became maximal between postnatal weeks 1 and 3. In the diencephalon, the highest densities of grains were found over neurons in the nucleus reticularis thalami and zona incerta at postnatal day 21; these levels declined slightly thereafter. The cells of the periventricular nucleus of the hypothalamus had high densities of grains as early as postnatal week 1 and continued to have high densities of grains in adult animals. These patterns of hybridization density parallelled the distribution of SOM-like immunoreactivity in the mouse brain. When PPSOM mRNA expression was examined in the cerebral cortices of mice that received lesions of the nucleus basalis of Meynert as neonates, a transient increase in the number of cells expressing PPSOM mRNA was observed in the frontoparietal cortex ipsilateral to the lesion at postnatal day 10, but not at postnatal day 30. Importantly, the density of grains over the individual cells was not altered in lesioned animals at these two ages.

Aging↗